Clostridium difficile is a spore-forming Gram-positive anaerobic bacillus, and is the leading cause of nosocomial diarrhea and colitis in the industrialized world with more than 300,000 cases of C. difficileassociated diarrhea (CDAD) reported each year in the United States alone. Broad spectrum antibiotic usage, hospitalization, advanced age and comorbidities increase the risk for acquiring CDAD. Symptoms result from the production of two potent C. difficile toxins (toxin A and toxin B). Studies with humans have shown that protection against disease and relapse correlates predominantly with the presence of serum IgG responses directed against toxin A and less strongly with toxin B. No vaccine effective at preventing C. difficile disease is currently commercially available, and measures to prevent CDAD through patient isolation and implementation of hand-hygiene and contact precautions have had variable and often limited success. We propose to develop a recombinant C. difficile protein vaccine by fusing the non-toxic receptor binding domain (RBD) of toxin A or toxin B with C. difficile flagellar proteins, FliC and FliD. The RBD of toxin A and toxin B have been shown to induce neutralizing antibodies in immunized mice. The C. difficile FliD and FliC are involved in adherence and gut colonization, and FliC is a potent Toll-like receptor (TLR) 5 ligand. TLRs are a family of pattern recognition receptors that recognize structural components shared by bacteria, fungi and viruses. TLRs when bound to their ligands such as flagellin can trigger innate responses as well as facilitate in the development of adaptive immunity. Several promising experimental vaccines have been tested with flagellin either as an antigen or as an adjuvant. It still remains to be determined whether anti-flagellin immune responses can prevent C. difficile colonization and whether activation through TLR signaling plays a significant role in human responses against a C. difficile toxin vaccine. We hypothesize that the incorporation of flagellar proteins and toxins in a vaccine could provide protection against colonization as well as disease progression. Key milestones will be to address whether the combination vaccine using toxins and flagellar proteins can exhibit robust immunogenicity in vaccinated mice, resulting in the production of toxin neutralizing antibodies, a correlate of vaccine efficacy, and anti-flagellar antibodies that can prevent colonization. Since C difficile is a mucosal pathogen, several routes of immunization that target the mucosal surface such as intrarectal, intranasal and transcutaneous will be compared to parenteral immunization in the presence of mucosal adjuvants such as heat labile enterotoxin, LT(r192g). The most promising vaccines will then be evaluated in challenge and protection studies. Challenge studies against multiple C. difficile strains in the mouse and the hamster model of bacterial infection will be performed to evaluate C difficile colonization and protection against CDAD.